Examining the role of TIMELESS in the Drosophila circadian clock

Drosophila melanogaster is a convenient model system for studying the molecular aspects of circadian clock function and the entrainment of the clock to environmental cycles. The current model for clock function involves the rhythmic cycling of two essential clock proteins, PERIOD (PER) and TIMELESS (TIM), which form a negative feedback loop inhibiting their own production by blocking circadian activators CLOCK (dCLK) and CYCLE (CYC). There are many regulatory steps in clock function that determine the timing of daily PER and TIM cycles, including cyclic transcription of mRNA, protein accumulation, nuclear translocation, and degradation. While PER has been extensively researched, the role of TIM has not. TIM is known to stabilize PER, allowing PER accumulation, and the rapid decrease of TIM after a light pulse is proposed to be an entrainment mechanism. But not only are there a number of untested assumptions about the role of TIM in feedback and nuclear localization, but the definitive experiments linking TIM destruction to light-mediated clock resetting have not been performed. We addressed several predictions about TIM through behavioral and in vitro tests, and found that TIM does not play a major role in transcription beyond allowing PER accumulation. We also determined that a long-held view that heterodimerization of PER and TIM is required for nuclear entry is not necessarily true. TIM, in particular, is nuclear in the absence of PER as long as nuclear export is blocked, indicating that it requires PER for nuclear retention. Overexpression of TIM has little effect on clock period. Such results confirm previous suggestions that PER is the state variable of the clock, and TIM enables PER cycling. To address the problem of whether TIM is necessary for entrainment, we tried to create a mutation in TIM that would knock out the TIM response to light. We have identified two lysine residues that are essential for full ubiquitylation, a necessary step in TIM degradation. Full analysis of this TIM mutant awaits its expression in transgenic fly lines. Overall, this research has led to a better understanding of TIM function in the Drosophila circadian clock.